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The human Alzheimer’s disease (AD) brain accumulates angiogenic markers but paradoxically, the cerebral microvasculature is reduced around Aß plaques. Here we demonstrate that angiogenesis is started near Aß plaques in both AD mouse models and human AD samples. However, endothelial cells express the molecular signature of non-productive angiogenesis (NPA) and accumulate, around Aß plaques, a tip cell marker and IB4 reactive vascular anomalies with reduced NOTCH activity. Notably, NPA induction by endothelial loss of presenilin, whose mutations cause familial AD and which activity has been shown to decrease with age, produced a similar vascular phenotype in the absence of Aß pathology. We also show that Aß plaque-associated NPA locally disassembles blood vessels, leaving behind vascular scars, and that microglial phagocytosis contributes to the local loss of endothelial cells. These results define the role of NPA and microglia in local blood vessel disassembly and highlight the vascular component of presenilin loss of function in AD., A.E.R.-N. was the recipient of a JdlC-F fellowship from the Spanish Ministry of Economy, Industry, and Competitiveness (MINEICO) (FJCI-2015-23708), M.I.A.-V., N.L.-U., and C.O.-d.S.L. were the recipient of an FPU fellowship from Spanish Ministry of Education, Culture, and Sport (respectively, FPU15/02898, FPU14-02115, and AP2010‐1598), and R.M.-D. was the recipient of a “Sara Borrell” fellowship from ISCIII (CD09/0007). Work was supported by grants to A.P. by the Spanish MINEICO, ISCIII, and FEDER (SAF2012‐33816, SAF2015‐64111‐R, RTI2018-096629-B-100, SAF2017-90794-REDT, and PIE13/0004), by the regional Government of Andalusia (“Proyectos de Excelencia”, P12‐CTS‐2138 and P12‐CTS‐2232) co-funded by CEC and FEDER funds, and by the “Ayuda de Biomedicina 2018”, Fundación Domingo Martínez; J.Vitorica: Instituto de Salud Carlos III (ISCiii) of Spain, co-financed by FEDER funds from European Union (PI18/01556) by La Marató-TV3 Foundation grant 20141431; by CIBERNED (CB06/05/0094); and by Junta de Andalucia Consejería de Economía y Conocimiento through grant US-1262734; A.G.: Instituto de Salud Carlos III (ISCiii) of Spain, co-financed by FEDER funds from European Union, through grant PI18/01557; and by Junta de Andalucia Consejería de Economía y Conocimiento through grants UMA18-FEDERJA-211 and P18-RT-2233 co-financed by Programa Operativo FEDER 2014-2020.

The detection of IgG aquaporin-4 antibodies in the serum of patients with Neuromyelitis optica (NMO) has dramatically improved the diagnosis of this disease and its distinction from multiple sclerosis. Recently, a group of patients have been described who have an NMO spectrum disorder (NMOsd) and who are seronegative for AQP4 antibodies but positive for IgG aquaporin-1 (AQP1) or myelin oligodendrocyte glycoprotein (MOG) antibodies. The purpose of this study was to determine whether AQP1 and MOG could be considered new biomarkers of this disease, and if point mutations in the gDNA of AQP4, AQP1 and MOG genes could be associated with the etiology of NMOsd. We evaluated the diagnostic capability of ELISA and cell-based assays (CBA), and analyzed their reliability, specificity, and sensitivity in detecting antibodies against these three proteins. The results showed that both assays can recognize these antigen proteins under appropriate conditions, but only anti-AQP4 antibodies, and not AQP1 or MOG, appears to be a clear biomarker for NMOsd. CBA is the best method for detecting these antibodies, and serum levels of AQP4 antibodies do not correlate with the progression of this disease. So far, the sequencing analysis has not revealed a genetic basis for the etiology of NMOsd, but a more extensive analysis is required before definitive conclusions can be drawn.

Activation of the epithelial-mesenchymal transition process (EMT) by which alveolar cells in human lung tissue undergo differentiation giving rise to a mesenchymal phenotype (fibroblast/miofibroblasts) has been well recognized as a key element in the origin of idiopathic pulmonary fibrosis (IPF). Here we analyzed expression of AQP1 in lung biopsies of patients diagnosed with IPF, and compared it to biopsies derived from patients with diverse lung pneumonies, such as hypersensitivity pneumonitis, sarcoidosis or normal lungs. Immunostaining for AQP1 showed a clear increment of AQP1 localized in the alveolar epithelium in biopsies from IPF patients alone. Moreover, to examine the possible participation of AQP1 in the pathophysiology of IPF, we evaluated its role in the pro-fibrotic transformation induced by transforming growth factor (TGF-β) in vitro. Human alveolar epithelial cells (A549), and fibroblasts derived from an IPF patient (LL29), or fibroblasts from healthy normal lung tissue (MRC-5), were treated with TGF-β, and levels of expression of AQP1, as well as those of E-cadherin, vimentin, α-SMA and collagen were analyzed by RT-qPCR, western blot and immunohistochemistry. An increase of AQP1 mRNA and protein after TGF-β treatment (4–72h) was observed either in A549 or IPF fibroblast-LL29 but not in MRC-5 fibroblasts. A gradual reduction of E-cadherin, and increased expression of vimentin, with no changes in α-SMA levels were observed in A549. Whereas in LL29 and MRC-5, TGF-β1 elicited a large production of collagen and α-SMA that was significantly greater in IPF fibroblast-LL29. Changes observed are consistent with activation of EMT by TGF-β, but whether modifications in AQP1 expression are responsible or independent events occurring at the same time is still unknown. Our results suggest that AQP1 plays a role in the pro-fibrotic TGF-β action and contributes to the etiology and pathophysiology of IPF. Understanding AQP1's role will help us comprehend the fate of this disease., This study has been supported by the grants PI12/01882 and PI16/00493 from the Spanish Ministry of Economy and Competitiveness, co-funded by ISCIII and FEDER funds, conceded to ME, and grants from Neumosur and SEPAR foundations given to JR.

Aquaporin-1 (AQP1) has been associated with tumor development. Here, we investigated how AQP1 may affect cell proliferation. The proliferative rate of adult carotid body (CB) cells, known to proliferate under chronic hypoxia, was analyzed in wild-type (AQP1(+/+) ) and knock out (AQP1(-/-) ) mice, maintained in normoxia or exposed to hypoxia while BrdU was administered. Fewer numbers of total BrdU(+) and TH-BrdU(+) cells were observed in AQP1(-/-) mice, indicating a role for AQP1 in CB proliferation. Then, by flow cytometry, cell cycle state and proliferation of cells overexpressing AQP1 were compared to those of wild-type cells. In the AQP1-overexpressing cells, we observed higher cell proliferation and percentages of cells in phases S and G2/M and fewer apoptotic cells after nocodazole treatment were detected by annexin V staining. Also in these cells, proteomic assays showed higher expression of cyclin D1 and E1 and microarray analysis revealed changes in many cell proliferation-related molecules, including, Zeb 2, Jun, NF-kβ, Cxcl9, Cxcl10, TNF, and the TNF receptor. Overall, our results indicate that the presence of AQP1 modifies the expression of key cell cycle proteins apparently related to increases in cell proliferation. This contributes to explaining the presence of AQP1 in many different tumors.

AQP3 has been correlated with higher transport of glycerol, increment of ATP content, and larger proliferation capacity. Recently, we described the gold(III) complex Auphen as a very selective and potent inhibitor of AQP3's glycerol permeability (Pgly ). Here we evaluated Auphen effect on the proliferation of various mammalian cell lines differing in AQP3 expression level: no expression (PC12), moderate (NIH/3T3) or high (A431) endogenous expression, cells stably expressing AQP3 (PC12-AQP3), and human HEK293T cells transiently transfected (HEK-AQP3) for AQP3 expression. Proliferation was evaluated in the absence or presence of Auphen (5 μM) by counting number of viable cells and analyzing 5-bromo-2'-deoxyuridine (BrdU) incorporation. Auphen reduced ≈50% the proliferation in A431 and PC12-AQP3, ≈15% in HEK-AQP3 and had no effect in PC12-wt and NIH/3T3. Strong arrest in the S-G2/M phases of the cell cycle, supported by analysis of cyclins (A, B1, D1, E) levels, was observed in AQP3-expressing cells treated with Auphen. Flow-cytometry of propidium iodide incorporation and measurements of mitochondrial dehydrogenases activity confirmed absence of cytotoxic effect of the drug. Functional studies evidenced ≈50% inhibition of A431 Pgly by Auphen, showing that the compound's antiproliferative effect correlates with its ability to inhibit AQP3 Pgly . Role of Cys-40 on AQP3 permeability blockage by Auphen was confirmed by analyzing the mutated protein (AQP3-Ser-40). Accordingly, cells transfected with mutated AQP3 gained resistance to the antiproliferative effect of Auphen. These results highlight an Auphen inhibitory effect on proliferation of cells expressing AQP3 and suggest a targeted therapeutic effect on carcinomas with large AQP3 expression.

Detection of IgG anti-Aquaporin-4 (AQP4) in serum of patients with Neuromyelitis optica syndrome disorders (NMOSD) has improved diagnosis of these processes and differentiation from Multiple sclerosis (MS). Recent findings also claim that a subgroup of patients with NMOSD, serum negative for IgG-anti-AQP4, present antibodies anti-AQP1 instead. Explore the presence of IgG-anti-AQP1 using a previously developed cell-based assay (CBA) highly sensitive to IgG-anti-AQP4. Serum of 205 patients diagnosed as NMOSD (8), multiple sclerosis (94), optic neuritis (39), idiopathic myelitis (29), other idiopathic demyelinating disorders of the central nervous system (9), other neurological diseases (18) and healthy controls (8), were used in a CBA over fixed HEK cells transfected with hAQP1-EGFP or hM23-AQP4-EGFP, treated with Triton X-100 and untreated. ELISA was also performed. Analysis of serum with our CBA indicated absence of anti-AQP1 antibodies, whereas in cells pretreated with detergent, noisy signal made reliable detection impossible. ELISA showed positive results in few serums. The low number of NMOSD serums included in our study reduces its power to conclude the specificity of AQP1 antibodies as new biomarkers of NMOSD. Our study does not sustain detection of anti-AQP1 in serum of NMOSD patients but further experiments are expected., Grants from “La Junta de Andalucía, Consejería de Innovación Ciencia y Empresa” (P08-CTS-03574) and Consejería de Salud (PI0298-2010), and from the “Instituto de Salud Carlos III” (Exp. PI12/01882) to Miriam Echevarría funded this work. We thank Genzyme Foundation in multiple sclerosis for giving to Miriam Echevarría one of their 2012 fellowships. We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

In addition to the extensive data demonstrating the importance of mammalian AQPs for the movement of water and some small solutes across the cell membrane, there is now a growing body of evidence indicating the involvement of these proteins in numerous cellular processes seemingly unrelated, at least some of them in a direct way, to their canonical function of water permeation. Here, we have presented a broad range of evidence demonstrating that these proteins have a role in cell proliferation by various different mechanisms, namely, by allowing fast cell volume regulation during cell division; by affecting progression of cell cycle and helping maintain the balance between proliferation and apoptosis, and by crosstalk with other cell membrane proteins or transcription factors that, in turn, modulate progression of the cell cycle or regulate biosynthesis pathways of cell structural components. In the end, however, after discussing all these data that strongly support a role for AQPs in the cell proliferation process, it remains impossible to conclude that all these other functions attributed to AQPs occur completely independently of their water permeability, and there is a need for new experiments designed specifically to address this interesting issue. Carlos III Health Institute (Ref. PI12/01882) Regional Department of Health (PI 0018-2014)

9 páginas, 4 figuras, 1 tabla., In this study, we explored the presence of aquaporins (AQPs), a family of membrane water channel proteins, in carotid body (CB) type I chemoreceptor cells. The CB is a polymodal chemoreceptor whose major function is to detect changes in arterial O2 tension to elicit hyperventilation during hypoxia. The CB has also been proposed to function as a systemic osmoreceptor, thus we hypothesized that the presence of AQPs in type I cell membrane may confer higher sensitivity to osmolarity changes and hence accelerate the activation of chemoreceptor cells. We detected the expression of AQP1, AQP7, and AQP8 in the CB and confirmed the location of AQP1 in type I cells. We have also shown that inhibition of AQP1 expression clearly reduced type I cell swelling after a hyposmotic shock, demonstrating that AQP1 has a major contribution in transmembrane water movement in these chemoreceptor cells. Interestingly, CB AQP1 expression levels change during postnatal development, increasing during the first postnatal weeks as the organ matures. In conclusion, in this study, we report the novel observation that AQPs are expressed in the CB. We also show that AQP1 mediates water transport across the cell membrane of type I cells, supporting the contribution of this protein to the osmoreception function of the CB., This work was funded by grants from the Ministry of Science and Innovation, “Instituto de Salud Carlos III” (PI PI060511 and CIBERNED), Junta de Andalucía (Consejería de Salud [exp. #0144 and #0192] and Consejería de Innovación Ciencia y Empresa [P08-CTS-03574]), and Marcelino Botín Foundation. AM Munoz-Cabello was supported by a predoctoral fellowship from the Spanish Ministry of Health. J. Villadiego is supported by a postdoctoral fellowship of Instituto de Salud Carlos III–Red de Terapia Celular (Spanish Ministry of Science and Innovation).

O(2) is essential for aerobic life, and the classic view is that it diffuses freely across the plasma membrane. However, measurements of O(2) permeability of lipid bilayers have indicated that it is much lower than previously thought, and therefore, the existence of membrane O(2) channels has been suggested. We hypothesized that, besides its role as a water channel, aquaporin-1 (AQP-1) could also work as an O(2) transporter, because this transmembrane protein appears to be CO(2)-permeable and is highly expressed in cells with rapid O(2) turnover (erythrocytes and microvessel endothelium). Here we show that in mammalian cells overexpressing AQP-1 and exposed to hypoxia, the loss of cytosolic O(2), as well as stabilization of the O(2)-dependent hypoxia-inducible transcription factor and expression of its target genes, is accelerated. In normoxic endothelial cells, knocking down AQP-1 produces induction of hypoxia-inducible genes. Moreover, lung AQP-1 is markedly up-regulated in animals exposed to hypoxia. These data suggest that AQP-1 has O(2) permeability and thus could facilitate O(2) diffusion across the cell membrane.

Abnormal AQP3 overexpression in tumor cells of different origins has been reported and a role for this enhanced AQP3 expression in cell proliferation and tumor processess has been indicated. To further understand the role AQP3 plays in cell proliferation we explore the effect that stable over expression of AQP3 produces over the proliferation rate and cell cycle of mammalian cells. The cell cycle was analyzed by flow cytometry with propidium iodide (PI) and the cell proliferation rate measured through cell counting and BrdU staining. Cells with overexpression of AQP3 (AQP3-o) showed higher proliferation rate and larger percentage of cells in phases S and G2/M, than wild type cells (wt). Evaluation of the cell response against arresting the cell cycle with Nocodazole showed that AQP3-o exhibited a less modified cell cycle pattern and lower Annexin V specific staining than wt, consistently with a higher resistance to apoptosis of AQP3-overexpressing cells. The cell volume and complexity were also larger in AQP3-o compared to wt cells. After transcriptomic analysis, RT-qPCR was performed to highlight key molecules implicated in cell proliferation which expression may be altered by overexpression of AQP3 and the comparative analysis between both type of cells showed significant changes in the expression of Zeb2, Jun, JunB, NF-kβ, Cxcl9, Cxcl10, TNF, and TNF receptors. We conclude that the role of AQP3 in cell proliferation seems to be connected to increments in the cell cycle turnover and changes in the expression levels of relevant genes for this process. Larger expression of AQP3 may confer to the cell a more tumor like phenotype and contributes to explain the presence of this protein in many different tumors.

Since specific proteins responsible for water transport (aquaporins, AQPs) have been identified in a great variety of tissues, we decided to study the presence of AQP3 in the gastrointestinal tract (GIT) of Wistar rats. Poly(A+) RNA was purified from the mucosa of the stomach, jejunum, ileum and colon, and gross detection of AQP3 mRNA was done by Northern blot analysis. In situ hybridization studies were carried out to precisely localize the distribution of this transcript. Sections of the different tissues were hybridized with @400-bp [35S]riboprobes. The results presented here demonstrate that AQP3 is expressed throughout the GIT, with its expression in the colon and ileum greater than that in the stomach. Immunohistochemistry experiments, using a polyclonal antibody against AQP3, revealed that AQP3 protein is present at the basolateral membrane of the epithelial cells lining the villus tip of the small intestine and colon. The finding of AQP3 in the intestinal epithelia strongly suggests that this protein functions as a pathway for water transport in this epithelium.

We have characterized the selectivity filter of the water channel aquaporin-1 (AQP1) of proximal straight tubules (PST), as an equivalent cylindrical structure with a diameter of approximately 4.5 A, where water molecules single file. We report here efforts to evaluate its length. PST were dissected from rabbit kidneys, held with pipettes in a chamber bathed in a buffered mannitol isosmotic solution (MBS, 295 mOsm/kg). Changes in tubule cell volume with time (dV/Adt), were monitored, on line, with an inverted microscope, a TV camera and an image processor. Osmotic permeability coefficients, Pos, and reflection coefficients (sigma s) were measured with several solutes: mannitol (M), raffinose (R), sucrose (S), glycerol (G), acetamide (A) and urea (U). For this purpose PST were suddenly exposed (in approximately 80 ms and for 20 s) to a hyperosmolality step (delta Cs) achieved by adding to MBS a delta Cs of 35 mOsm/kg of either R, S, M, G, A or U. Cells shrunk within 500 ms of t = 0 to their osmometric volume and remained shrunk for the 20 s of the delta Cs. Pos was measured from the shrinking curves; Pos = 3000 +/- 25 microns/s with either R, S, M, G, A or U. This procedure also allowed to calculate sigma s; sigma s = 1.00 for R, S, M, G, A and U, indicating that these solutes do not penetrate the water channel. In contrast, the shrinking curves produced by a delta Cs = 35 mOsm/kg formamide (F) were 1/5th to 1/6th slower and smaller (subosmometric) than those produced by a delta Cs = 35 mOsm/kg of R, S, M, G, A or U. Furthermore, with F, cells did not remain shrunk. They recovered their original volume within 3 s. Pos (measured with F) is denoted as Pos*; Pos* = 480 +/- 30 microns/s. sigma s, formamide (denoted sigma sp) = 0.16 +/- 0.01. Use of sigma sp and Pos* values in Hill's equations for the bimodal theory of osmosis leads to n = 2-3, n being the number of water molecules single filing within the channel selectivity filter, whose length must lie within 6 to 9 A, a value lower than previous values calculated from the Pos/Pd* ratio.

Overexpression of cell membrane aquaporins (AQPs) has recently been associated with tumor formation, particularly with angiogenesis, cell migration and proliferation. Additionally, the hypoxia inducible factor (HIF) family has been extensively implicated in tumor growth and recent studies evidence interplay between AQP expression and HIF stability. Therefore, we decided to explore the effect that AQP overexpression has on the long-term stability of HIF-2α in PC12 cells exposed to chronic hypoxia, characteristic of a growing tumor. HIF-2α levels were analyzed in five PC12 clones with stable overexpression of different proteins (AQP1, AQP3, AQP5, G6PD, and GDNF), in PC12 transiently expressing G6PD or Kv4.2, and in wild-type PC12 cells. Overexpression of AQP1, 3 or 5 in PC12 cells (o-AQP-c) prevented the HIF-2α down-expression otherwise observed, after 16h at 1% O2, in wt-PC12 and in PC12 overexpressing non-AQP proteins. Longer HIF-2α stability was also observed in o-AQP-c exposed to cobalt chloride or dimethyloxallyl glycine. Normal proteasome activity was confirmed in all clones analyzed. Levels of HIF target genes (PHD2 and 3, VEGF, and PGK1) were 2-4 fold higher in hypoxic o-AQP-c than in wt-PC12 cells, and morphological changes in colony shape together with higher cell proliferation rates were observed in all o-AQP-c. Interestingly, analysis of PHD levels under normoxia revealed lower (50%) PHD3 expression in o-AQP-c than in controls. Our results indicate that AQP overexpression in PC12 cells prolongs HIF-2α stability during chronic hypoxia, leading to higher level of induction of its target genes and likely conferring to these cells a more tumor-like phenotype. © 2013 Elsevier B.V., This work was funded by grants from the “Instituto de Salud Carlos III” PS09/00605, and by grants from “La Junta de Andalucía”, Consejería de Salud (PI0298-2010) and Consejería de Innovación Ciencia y Empresa (P08-CTS-03574). AGC was supported by a predoctoral fellowship from “Junta de Andalucía, Consejería de Innovación Ciencia y Empresa”, Spain and IAR was supported by a predoctoral fellowship from Spanish Ministry of Science and Innovation.

The aquaporins are a family of water channels expressed in several water-transporting tissues, including the kidney. We have used a peptide-derived, affinity-purified polyclonal antibody to aquaporin-3 (AQP-3) to investigate its localization and regulation in the kidney. Immunoblotting experiments showed expression in both renal cortex and medulla, with greatest expression in the base of the inner medulla. Subcellular fractionation of membranes, using progressively higher centrifugation speeds, revealed that AQP-3 is present predominantly in the 4,000 and 17,000 g pellets and, in contrast to AQP-2, is virtually absent in the high-speed (200,000 g) pellet that contains small intracellular vesicles. Immunocytochemistry and immunofluorescence studies revealed that labeling is restricted to the cortical, outer medullary, and inner medullary collecting ducts. Within the collecting duct, principal cells were labeled, whereas intercalated cells were unlabeled. Consistent with previous immunofluorescence studies (K. Ishibashi, S. Sasaki, K. Fushimi, S. Uchida, M. Kuwahara, H. Saito, T. Furukawa, K. Nakajima, Y. Yamaguchi, T. Gojobori, and F. Marumo. Proc. Natl. Acad. Sci. USA 91: 6269-6273, 1994; T. Ma, A. Frigeri, H. Hasegawa, and A. S. Verkman. J. Biol. Chem. 269: 21845-21849, 1994), the labeling was confined to the basolateral domain. Immunoelectron microscopy, using the immunogold technique in ultrathin cryosections, demonstrated a predominant labeling of the basolateral plasma membranes. In contrast to previous findings with AQP-2, there was only limited AQP-3 labeling of intracellular vesicles, suggesting that this water channel is not regulated acutely through vesicular trafficking. Immunoblotting studies revealed that thirsting of rats for 48 h approximately doubled the amount of AQP-3 protein in the inner medulla. These studies are consistent with a role for AQP-3 in osmotically driven water absorption across the collecting duct epithelium and suggest that the expression of AQP-3 is regulated on a long-term basis.

Aquaporins (AQPs) are membrane channels that conduct water and small solutes such as glycerol and are involved in many physiological functions. Aquaporin-based modulator drugs are predicted to be of broad potential utility in the treatment of several diseases. Until today few AQP inhibitors have been described as suitable candidates for clinical development. Here we report on the potent inhibition of AQP3 channels by gold(III) complexes screened on human red blood cells (hRBC) and AQP3-transfected PC12 cells by a stopped-flow method. Among the various metal compounds tested, Auphen is the most active on AQP3 (IC 50 = 0.8±0.08 μM in hRBC). Interestingly, the compound poorly affects the water permeability of AQP1. The mechanism of gold inhibition is related to the ability of Au(III) to interact with sulphydryls groups of proteins such as the thiolates of cysteine residues. Additional DFT and modeling studies on possible gold compound/AQP adducts provide a tentative description of the system at a molecular level. The mapping of the periplasmic surface of an homology model of human AQP3 evidenced the thiol group of Cys40 as a likely candidate for binding to gold(III) complexes. Moreover, the investigation of non-covalent binding of Au complexes by docking approaches revealed their preferential binding to AQP3 with respect to AQP1. The high selectivity and low concentration dependent inhibitory effect of Auphen (in the nanomolar range) together with its high water solubility makes the compound a suitable drug lead for future in vivo studies. These results may present novel metal-based scaffolds for AQP drug development. © 2012 Martins et al., This work was supported from Fundação para a Ciência e a Tecnologia, Portugal, through a Ph.D. fellowship to APM (SFRH/BD/65046/2009). A. Casini thanks the University of Groningen (Rosalind Franklin fellowship). The authors thank COST CM0902 for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Intrastriatal transplantation of dopaminergic carotid body (CB) cells ameliorates parkinsonism in animal models and, with less efficacy, in Parkinson's disease patients. CB-based cell therapy was initially proposed because of its high dopamine content. However, later studies suggested that its beneficial effect might be due to a trophic action exerted on nigrostriatal neurons. Compatible with this concept are the high levels of neurotrophic factors encountered in CB cells. To test experimentally this idea, unilateral striatal transplants were performed with a sham graft in the contralateral striatum, as a robust internal control. Thereafter, the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6, -tetrahydropyridine was injected during 3 months. CB grafts protected from degeneration ipsilateral nigral dopaminergic neurons projecting to the transplant in a dose-dependent manner regarding size and glial cell line-derived neurotrophic factor expression. Grafts performed at different times after the onset of the neurotoxic treatment demonstrated with histological and behavioral methods protection and repair of the nigrostriatal pathway by CB transplants. This study provides a mechanistic explanation for the action of CB transplants on parkinsonian models. It should also help to improve cell therapy approaches to Parkinson's disease. © 2013 Elsevier Inc., This study was supported by grants from the Spanish Government (FIS, Red TERCEL, CIBERNED, MEC, and MICINN), the Andalusian Government (Excellence projects) and the Marcelino Botín Foundation.

To test the hypothesis that renal tissue contains multiple distinct water channels, mRNA prepared from either cortex, medulla, or papilla of rat kidney was injected into Xenopus oocytes. The osmotic water permeability (Pf) of oocytes injected with either 50 nl of water or 50 nl of renal mRNA (1 microgram/microliter) was measured 4 d after the injection. Pf was calculated from the rate of volume increase on exposure to hyposmotic medium. Injection of each renal mRNA preparation increased the oocyte Pf. This expressed water permeability was inhibited by p-chloromercuriphenylsulfonate and had a low energy of activation, consistent with the expression of water channels. The coinjection of an antisense oligonucleotide for CHIP28 protein, at an assumed > 100-fold molar excess, with either cortex, medulla, or papilla mRNA reduced the expression of the water permeability by approximately 70, 100, and 30%, respectively. Exposure of the oocyte to cAMP for 1 h resulted in a further increase in Pf only in oocytes injected with medulla mRNA. This cAMP activation was not altered by the CHIP28 antisense oligonucleotide. These results suggest that multiple distinct water channels were expressed in oocytes injected with mRNA obtained from sections of rat kidney: (a) CHIP28 water channels in cortex and medulla, (b) cAMP-activated water channels in medulla, and (c) cAMP-insensitive water channels in papilla.

Oxidative damage to dopaminergic nigrostriatal (DNS) neurons plays a central role in the pathogenesis of Parkinson's disease (PD). Glucose-6-phosphate dehydrogenase (G6PD) is a key cytoprotective enzyme that provides NADPH, the major source of the reducing equivalents of a cell. Mutations of this enzyme are the most common enzymopathies worldwide. We have studiedin vivothe role of G6PD overexpressed specifically in the DNS pathway and show that the increase of G6PD activity in the soma and axon terminals of DNS neurons, separately from other neurons or glial cells, protects them from parkinsonism. Analysis of DNS neurons by histological, neurochemical, and functional methods showed that even a moderate increase of G6PD activity rendered transgenic mice more resistant than control littermates to the toxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The neuroprotective action of G6PD was also observed in aged animals despite that they had a greater susceptibility to MPTP. Therefore, overexpression of G6PD in dopaminergic neurons or pharmacological activation of the native enzyme should be considered as potential therapeutic strategies to PD.

Aquaporins (AQPS) are transmembrane water channels poorly investigated in birds. Using degenerated primers and RT-PCR, we identified in kidney and gastrointestinal tract of Hubbard chickens (Gallus gallus) three fragments, corresponding to ck-AQP2, ck-AQP4, and ck-AQP5 mRNAs. Comparison of nucleotide ck-AQPs sequences to their rat and human orthologues revealed an overall identity of 75-90%. Expression in the renal and gastrointestinal systems of the three ck-AQPs mRNA was analysed by Northern assays. Transcript of ck-AQP2 was only identified in kidney. ck-AQP4 mRNA was highly expressed in brain, and to a lesser extent in kidney and stomach. ck-AQP5 mRNA was found in jejunum and ileum, and to a lesser extent in colon and lung. In situ hybridisation showed ck-AQP5 mRNA in the crypt cells of jejunum, ileum and colon, whereas it was absent from the cells lining the villi. Levels of ck-AQP5 mRNA (analyzed by Northern and in situ hybridisation assays) and protein (analysed by immunohistochemistry) decreased from the jejunum to the colon. This work confirmed the presence of AQPs in chicken, and showed that chicken and mammalian AQPs share a high degree of similarity in nucleotide sequence and tissue distribution.

We studied the participation of adrenal medulla (AM) chromaffin cells in hypercapnic chemotransduction. Using amperometric recordings, we measured catecholamine (CAT) secretion from cells in AM slices of neonatal and adult rats perfused with solutions bubbled with different concentrations of CO2. The secretory activity augmented from 1.74 ± 0.19 pC/min at 5% CO2to 6.36 ± 0.77 pC/min at 10% CO2. This response to CO2was dose dependent and appeared without changes in extracellular pH, although it was paralleled by a drop in intracellular pH. Responsiveness to hypercapnia was higher in neonatal than in adult slices. The secretory response to hypercapnia required extracellular Ca2+influx. Both the CO2-induced internal pH drop and increase in CAT secretion were markedly diminished by methazolamide (2 μm), a membrane-permeant carbonic anhydrase (CA) inhibitor. We detected the presence of two CA isoforms (CAI and CAII) in neonatal AM slices byin situhybridization and real-time PCR. The expression of these enzymes decreased in adult AM together with the disappearance of responsiveness to CO2. In patch-clamped chromaffin cells, hypercapnia elicited a depolarizing receptor potential, which led to action potential firing, extracellular Ca2+influx, and CAT secretion. This receptor potential (inhibited by methazolamide) was primarily attributable to activation of a resting cationic conductance. In addition, voltage-gated K+current amplitude was also decreased by high CO2. The CO2-sensing properties of chromaffin cells may be of physiologic relevance, particularly for the adaptation of neonates to extrauterine life, before complete maturation of peripheral and central chemoreceptors.

We studied the mechanisms underlying long-term functional recovery of hemiparkinsonian rats grafted intrastriatally with carotid body (CB) cell aggregates. Amelioration of their motor syndrome is a result of the trophic actions of these grafts on the remaining ipsilateral substantia nigra neurons rather than of the release of dopamine from the CB grafts. The grafts maintain a stable morphological appearance and differentiated cell phenotype for the duration of the life of the host. Adult CB expresses high levels of glial cell line-derived neurotrophic factor (GDNF) and the multicomponent GDNF receptor complex. These properties may contribute to the trophic actions of the CB transplants on nigrostriatal neurons and to their extraordinary longevity. We show that CB glomus cells, although highly dopaminergic, are protected from dopamine-mediated oxidative damage because of the absence of the high-affinity dopamine transporter. Thus, intrastriatal CB grafts are uniquely suited for long-term delivery of trophic factors capable of promoting restoration of the nigrostriatal pathway.

Malpighian tubules (MT) of Rhodnius prolixus transport fluid at very high rates. To identify whether aquaporins (AQPs) are present in the MT of R. prolixus, total ribonucleic acid (RNA) was isolated from MT and used in a reverse transcription, polymerase chain reaction (RT-PCR), with two degenerate primers to highly conserved regions of the members of the AQPs family. A deoxyribonucleic acid (DNA) fragment of 370 bp was amplified; its sequence revealed a novel protein, representing a new member of the major intrinsic protein (MIP) family. The complementary DNA (cDNA) sequence of this new MIP protein was cloned by using RNA from MT and the rapid amplification of cDNA ends (RACE) technique. The cDNA had 1133 bp and the largest open reading frame coded for a protein of 286 amino acids, named R. prolixus major intrinsic protein (Rp-MIP). The hydrophobicity profile of the amino acid sequence predicts six transmembrane domains. Northern blot analysis of MT RNA showed a single transcript of about 1–1.3 kb for Rp-MIP. RT-PCR of single isolated MT and in situ hybridization analysis showed Rp-MIP transcripts in both proximal and distal segments. Expression of Rp-MIP in Xenopus laevis oocytes doubled the osmotic water permeability Pf, indicating that Rp-MIP may function as an aquaporin protein in the MT of the insect and thus may participate in urine formation in R. prolixus.

We have performed a detailed in situ hybridization study of the distribution of aquaporin-4 messenger RNA in the CNS. Contrary to expectation, we demonstrate that aquaporin-4 is ubiquitously expressed in the CNS. Strong hybridization labeling was detected in multiple olfactory areas, cortical cells, medial habenular nucleus, bed nucleus of the stria terminalis, tenia tecta, pial surface, pontine nucleus, hippocampal formation and multiple thalamic and hypothalamic areas. A low but significant hybridization signal was found, among others, in the choroid plexus of the lateral ventricles, ependymal cells, dorsal raphe and cerebellum. Overall, a preferential distribution of aquaporin-4 messenger RNA-expressing cells was evident in numerous periventricular organs. From the distribution study, the presence of aquaporin-4 messenger RNA-expressing cells in neuronal layers was evident in neuronal layers including the CA1 -CA3 hippocampal pyramidal cells, granular dentate cells and cortical cells. Further evidence of neuronal expression comes from the semicircular arrangement of aquaporin-4 messenger RNA-expressing cells in the bed nucleus of the stria terminalis and medial habenular nucleus exhibiting Nissl-stained morphological features typical of neurons. Combined glial fibrillary acidic protein immunohistochemistry and aquaporin-4 messenger RNA in situ hybridization demonstrated that aquaporin-4 messenger RNA is expressed by glial fibrillary acidic protein-lacking cells. We conclude that aquaporin-4 messenger RNA is present in a collection of structures typically involved in the regulation of water and sodium intake and that aquaporin-4 water channels could be the osmosensor mechanism responsible for detecting changes in cell volume by these cells.

The chromosome location of the gene encoding aquaporin 3 (AQP3), which functions as a channel for water and small polar solutes in the basolateral membrane of the collecting duct of the kidney, was determined. In situ hybridization on metaphase chromosomes allowed the assignment of human AQP3 to chromosome 9p21→p12.

The terminal part of the inner medullary collecting duct exhibits a high degree of water permeability that is independent of increased intracellular cAMP and not accounted for by the activity of the known renal epithelial water channels CHIP28 (28-kDa channel-forming integral protein) and WCH-CD (collecting duct water channel protein). Starting with rat kidney papilla mRNA, reverse transcription PCR was performed with degenerate primers assuming that the putative channel would be a member of the major intrinsic protein (MIP) family of proteins. A cDNA fragment was identified and used to screen a rat kidney cDNA library. A 1.9-kb cDNA clone was isolated. The open reading frame of 876 bp coded for a protein of 292 amino acids (M(r), 31,431). Aquaporin 3 (AQP3; 31.4-kDa water channel protein) is a newly discovered member of the MIP family. Northern blot analysis showed a single transcript for AQP3 of approximately 1.9 kb present in the renal medulla, predominantly in the inner medulla. With in situ hybridization, abundant message was found in the cells of the medullary collecting ducts. Injection of the complementary RNA of AQP3 into Xenopus oocytes markedly increased the osmotic water permeability. This permeability had an energy of activation of 3.0 kcal/mol (1 cal = 4.184 J), it was fully blocked by 1 mM p-chloromercuriphenylsulfonate, and this inhibition was reversed by 5 mM dithiothreitol. cAMP did not increase this water permeability. AQP3 did not permit passage of monovalent ions (Na, K, Cl); however, it is slightly permeable to urea. The present study demonstrates the existence of an additional water channel, AQP3, in epithelial cells of the medullary collecting duct.

Aquaporin-1 (AQP1) is a water channel that is highly expressed in tissues with rapid O2 transport. It has been reported that this protein contributes to gas permeation (CO2, NO and O2) through the plasma membrane. We show that hypoxia increases Aqp1 mRNA and protein levels in tissues, namely mouse brain and lung, and in cultured cells, the 9L glioma cell line. Stopped-flow light-scattering experiments confirmed an increase in the water permeability of 9L cells exposed to hypoxia, supporting the view that hypoxic Aqp1 up-regulation has a functional role. To investigate the molecular mechanisms underlying this regulatory process, transcriptional regulation was studied by transient transfections of mouse endothelial cells with a 1297 bp 5′ proximal Aqp1 promoter-luciferase construct. Incubation in hypoxia produced a dose- and time-dependent induction of luciferase activity that was also obtained after treatments with hypoxia mimetics (DMOG and CoCl2) and by overexpressing stabilized mutated forms of HIF-1α. Single mutations or full deletions of the three putative HIF binding domains present in the Aqp1 promoter partially reduced its responsiveness to hypoxia, and transfection with Hif-1α siRNA decreased the in vitro hypoxia induction of Aqp1 mRNA and protein levels. Our results indicate that HIF-1α participates in the hypoxic induction of AQP1. However, we also demonstrate that the activation of Aqp1 promoter by hypoxia is complex and multifactorial and suggest that besides HIF-1α other transcription factors might contribute to this regulatory process. These data provide a conceptual framework to support future research on the involvement of AQP1 in a range of pathophysiological conditions, including edema, tumor growth, and respiratory diseases., This work was funded by grants from the “Instituto de Salud Carlos III” PI PI060511 and PS09/00605, and by grants from “La Junta de Andalucía”, Consejería de Salud (exp. # 0144 and # 0192) and Consejería de Innovación Ciencia y Empresa (P08-CTS-03574). IAR was supported by a predoctoral fellowship from the Spanish Ministry of Science and Innovation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We studied the regulation of glucose-6-phosphate dehydrogenase (G6PD) gene expression by chronic hypoxia. G6PD mRNA level and activity were increased in PC12 cells by hypoxia in a dose- and time-dependent manner. Cobalt chloride and dimethyloxalylglycine, which can mimic hypoxia, also activated G6PD gene expression. Interestingly, hypoxia-induced G6PD expression followed a time course much slower than that of phosphoglycerate kinase 1 (PGK1), a hypoxia-inducible factor (HIF)-dependent glycolytic enzyme. Hypoxic-G6PD induction was almost negligible in non-excitable Buffalo rat liver cells, although in these cells PGK1 was strongly upregulated by low PO2. Furthermore, G6PD but not PGK1 induction was blocked by the antioxidants glutathione and N-acetylcysteine. These results suggest the dependence of G6PD gene expression on HIF and intracellular redox status and the differential hypoxic regulation of glucose-metabolizing enzymes.

Three aquaporins (AQP) are present in the membrane of the principal collecting duct cells. On the apical side, the levels of AQP2 protein are increased in response to both arginine vasopressin and water deprivation. However, whether this change parallels changes in the abundance of AQP3 and AQP4 in the basolateral membrane is less well known. This study evaluates the effect of either dehydration or water loading on the rat kidney mRNA expression of AQP2, AQP3, and AQP4. Poly(A + )RNA was prepared from renal cortex and medulla of control, water-deprived, well hydrated, and water-deprived rats treated with OPC31260, a V2 receptor antagonist. Northern blots were done and mRNA levels were quantified using a PhosphorImager system. Relative to control, water deprivation increased the expression of cortical AQP2, -3, and -4, whereas water loading decreased the cortical and medullar expression of AQP2, -3, and -4. Therefore, in addition to AQP2 and -3, AQP4 expression is also regulated by water intake. Treatment with OPC31260 (40 mg/kg of weight per d) inhibited up to 20 to 30% the upregulation of AQP-mRNA induced by water deprivation. Blood values of arginine vasopressin and aldosterone were significantly increased by water deprivation, whereas they were unchanged by water overloading. Taken together, these results indicate that renal AQP2, -3, and -4 expression is regulated in a coordinated manner. Simultaneous up- or downregulation of the three transcripts occurred upon either water deprivation or water loading of animals, respectively. However, the signaling mechanism for the two longterm adaptive processes may be different, and, in addition to arginine vasopressin, other factors may be involved in the transcriptional regulatory processes.